Membrane fusion is the key molecular event during the entry of enveloped viruses into cells, but it is not understood how the activity of fusion proteins is coordinated to catalyze this kinetically unfavorable process. Knowledge of the kinetics of the fusion reaction is largely derived from studies where many fusion events are observed simultaneously, thereby making a straightforward quantitative Analysis difficult. The objective of this proposal is to study viral membrane fusion by a fundamentally newstrategy: reconstituting viral fusion in vitro with only the bare minimum of molecular components and monitoring the kinetics of individualfusion events. The resulting 'molecular movies' will allow usto dissect the reaction kinetics at a level of detail inaccessible to conventional ensemble experiments. As a model system, the hemagglutinin (HA) fusion machinery of influenza virus will be used. HA- containing virosomes will be labeled with different fluorescent probes and fluorescence microscopy used to monitor the kinetics effusion and hemifusion of the individual particles with a planar, supported bilayer. The Specific Aims are: 1) to determine the kinetics of the transitions between the various bilayer fusion intermediates and establish the roles of the different hemifusion states, and 2) to determine the molecular nature of the cooperative behavior in HA-mediated fusion. The long-term goal of the proposed work is to combine the kinetic information as obtained from single- particle studies with known structural and biochemical properties into one complete molecular picture that accurately describes the mechanism of membrane fusion in influenza infection. Furthermore, the proposed single-particle techniques will be directly applicable to studying molecular mechanisms of membrane fusion in other systems. Public health relevance: Enveloped viruses invade cells by merging their membrane with the lipid bilayer surrounding the target cell. A detailed understanding of the molecular details of this fusion process is a necessary tool in the identification of drug targets and the rational design of therapeutics. The proposed development and use of single-particle imaging techniques to study the fusion mechanism of influenza will lead to a more complete and dynamic picture of the various pathways involved.